Small-sized apertures find use in a variety of applications including scanning microscopes, ink-jet nozzles, and micromechanical systems. Small apertures are often made using microfabrication techniques. Microfabrication techniques and applications may be found, for example, in U.S. Pat. No. 5,517,280 to Quate, U.S. Pat. No. 5,858,256 to Minne et al., U.S. Pat. No. 5,943,075 to Lee et al., and U.S. Pat. No. 6,219,015 to Bloom. All patents, both supra and infra, are hereby incorporated by reference in their entirety.
For example, microfabrication techniques are used in the manufacture of semiconductor components. The most common microfabrication technique is photolithography, in which photoresist layers on a substrate are exposed to radiation and so made more resistant to chemical processes that are to be applied later to the substrate. In this way, specific regions of the substrate may be removed or altered by chemical action while neighboring regions are not removed or altered. A photoresist pattern may be formed by a photomask made from masking material placed in a pattern on a substrate by a stamp, by sputtering, by optical methods, or by other means. Typically, a photomask is used to block irradiation of some regions while allowing other regions of the substrate surface to be irradiated by light in order to produce a desired pattern of resistant and susceptible regions. Features of such patterns produced by ultraviolet radiation may be as small as about 0.5 μm. However, features smaller than about 0.5 μm cannot generally be formed by these techniques.
Artificial lipid bilayer membranes (ALBMs) are membranes formed with phospholipids and sometimes solvents that mimic many of the properties of biological membranes. ALBMs may be formed across small apertures (that are nevertheless typically larger than cells or cellular organelles). Solvent-containing membranes, which differ from living membranes by including solvents such as decane or hexadecane, may be formed across apertures in hydrophobic materials such as polytetrafluoroethylene (Teflon®), polystyrene, and polyethylene. Typically, such apertures must first be treated with a solvent before application of a mixture of solvent and phospholipid to form a membrane. Preferably, ALBMs may be formed with little or no solvent from phospholipid monolayers by Langmuir-Blodgett techniques. See, for example Montal and Mueller, Proc. Natl. Acad. Sci. USA. 69:3561-3566 (1972); Montal, Meth. Enzymol. 32:545-556 (1974); and Lindstrom et al., J. Biol. Chem. 255:8340-8350 (1980).
Solvent-free ALBMs are very thin, being approximately as thick as the length of two phospholipid molecules laid end-to-end. Even solvent-containing artificial lipid bilayers, which may have a layer of solvent between the two phospholipid leaflets, are extremely thin. One method of determining whether a bilayer has been formed is to observe the disappearance of Newton's rings (colored patterns caused by interference between light reflected from each external surface of a thinning lipid membrane) as the membrane thins to a thickness less than that of a wavelength of visible light.
ALBMs are not as stable as membranes found in living cells. In living organisms, lipid bilayers form structures that may be as small as only a few nanometers to a few tens of nanometers in size. Although thin, artificial lipid bilayers usually have much more surface area than the bilayer membranes that surround living cells and intracellular organelles. In addition, ALBMs are formed across artificial apertures, which may have discontinuities and defects at the edges and surfaces and which are typically formed by manual processes which preclude uniformity of manufacture. Thus, ALBMs quite often rupture spontaneously within a few seconds or minutes after formation, possibly due to their relatively large surface area, imperfections in the apertures, and their lack of the small molecules, proteins and sugars found in living membranes which may enhance the membrane stability.
Biosensors are devices that utilize biomolecules to recognize target molecules in a highly specific manner. Biosensors are used commercially for sensing glucose, and are used in research and industrial applications to detect other molecules, often at extremely low concentrations. For example, enzymes are used to detect the presence of molecules that serve as substrates for a chemical reaction catalyzed by the enzyme. Ligand receptors are exquisitely sensitive to target molecules, often responding to the binding of a single target molecule. Ion channels may respond to ligand binding, to small voltage changes, to mechanical stress, or to other signals and so are also useful for detecting changes in their environment.
The biomolecules used as detectors in biosensors are typically membrane-bound or membrane-associated molecules derived from plant or animal cells. The activity of such molecules is quite often critically dependent upon the membrane with which they are associated or to which they are bound. In addition, where a biosensor depends upon a membrane-bound biomolecule as a sensor element, instability or rupture of the membranes destroys the sensor element. Accordingly, methods and devices for creating suitable membranes for use in biosensors are needed.